A betacyanin-loaded composite hydrogel, and a preparation method and application thereof

Abstract

This invention belongs to the field of biopolymer material preparation technology, and discloses a betaine-loaded composite hydrogel, its preparation method and application, including the following steps: (1) heat-treating an ovalbumin solution under acidic conditions to induce its self-assembly into an ovalbumin amyloid fiber dispersion; (2) mixing the ovalbumin amyloid fiber dispersion with a gelatin solution, stirring and dissolving to obtain a matrix mixture; (3) adding betaine and transglutaminase to the obtained matrix mixture, reacting at 37~55℃ for 0.5~3h, and then cooling and fixing to obtain the betaine-loaded composite hydrogel. This invention prepares a composite hydrogel with a dense three-dimensional network structure and high mechanical strength, while improving the photothermal stability of betaine. The obtained composite hydrogel achieves efficient encapsulation and sustained-release control of betaine at a low addition amount, and can be widely used in functional foods and biopharmaceutical industries.

Description

Technical Field

[0001] This invention belongs to the technical field of biopolymer materials and functional delivery systems, specifically relating to a composite hydrogel based on glutamine transaminase-modified gelatin and ovalbumin amyloid fibers and its application in betalain delivery. Background Technology

[0002] Betanin, a natural functional ingredient with highly effective antioxidant and free radical scavenging properties, is widely used in food coloring, nutritional fortification, and pharmaceuticals. However, betanin is extremely sensitive to light, heat, oxygen, and pH conditions, and is highly susceptible to oxidative degradation during food processing, storage, and human gastrointestinal digestion. This not only reduces its color stability but also severely limits its bioactivity. Therefore, developing efficient encapsulation and delivery systems to improve the processing stability and bioavailability of betanin is of great significance.

[0003] Gelatin, with its excellent biocompatibility and gelling properties, is an ideal food delivery matrix. Ovalbumin amyloid filaments (OAFs), as a novel protein nanomaterial, possess high aspect ratios, abundant hydrophobic microdomains, and excellent structural rigidity, and are considered highly promising nano-reinforcing phases. Through transglutaminase (TGase)-mediated enzymatic cross-linking, covalent bonds can be induced between protein molecules under mild conditions to construct a composite network system with both physical and chemical reinforcement. This composite material can utilize the nano-confining effect of OAFs and the tight cross-linking of TGase to provide multiple barriers for betaine protection.

[0004] Currently, traditional hydrogel carriers used for delivering betalains generally suffer from low mechanical strength, poor thermal stability, and premature release due to structural loosening in simulated gastric environments (low pH). Furthermore, existing technologies mostly employ physical blending methods, which have limited photothermal shielding capabilities within the internal network and often fail to balance biocompatibility and efficient delivery performance. While some studies have used chemical cross-linking agents, these pose potential toxic residues and do not meet the green and healthy requirements of the food industry. Therefore, there is an urgent need for a composite hydrogel system based on pure natural protein components, with a mild preparation process and excellent stabilization protection, to achieve efficient encapsulation and targeted delivery of betalains. Summary of the Invention

[0005] To address the aforementioned problems, this invention proposes a method for preparing betalain-loaded composite hydrogels using transglutaminase (TGase)-mediated enzymatic cross-linking combined with protein nanofiber reinforcement technology. This method utilizes ovalbumin amyloid fibers as a nanoskeleton, coupled with the covalent reinforcement effect of TGase, significantly improving the encapsulation efficiency and physicochemical stability of betalain in the gelatin matrix.

[0006] The present invention adopts the following technical solution:

[0007] A method for preparing a betaine-loaded composite hydrogel includes the following steps:

[0008] (1) The ovalbumin solution was heat-treated under acidic conditions to induce its self-assembly into an ovalbumin amyloid filament (OAFs) dispersion;

[0009] (2) Mix the ovalbumin amyloid fiber dispersion with the gelatin solution and stir to dissolve to obtain a matrix mixture;

[0010] (3) Add betaine and glutamine transaminase (TGase) to the obtained matrix mixture, react at 37~55℃ for 0.5~3h, and then cool and fix to obtain betaine-loaded composite hydrogel.

[0011] Preferably, in step (2), the mass ratio of gelatin to ovalbumin amyloid dispersion is 10:1-1:1, the concentration of ovalbumin amyloid fiber dispersion is 0.5~2.0 wt%, and the concentration of gelatin solution is 0.05~0.15 g / mL.

[0012] Preferably, the mass ratio of gelatin to ovalbumin amyloid cellulose dispersion in step (2) is 5:1-5:4; and the pH of ovalbumin amyloid cellulose dispersion is 2.0±0.5.

[0013] Preferably, the ovalbumin amyloid cellulose is prepared by the following method: adjusting the pH of an ovalbumin solution with a concentration of 1.0~2.0 wt% to 1.5~2.5 with hydrochloric acid, and stirring at a constant temperature of 80~95℃ for 12~24h.

[0014] Preferably, the amount of transglutaminase added in step (3) is 10~50 U / g, based on the weight of gelatin.

[0015] Preferably, the amount of transglutaminase added in step (3) is 20~40 U / g, based on the weight of gelatin.

[0016] Preferably, the amount of betalain added is 0.1 to 0.5 wt% of the gelatin mass.

[0017] Preferably, the stirring and dissolving conditions in step (2) are mechanical stirring at 500-1000 rpm for 15-30 min at 40-60℃; the reaction temperature in step (3) is 45-50℃ and the reaction time is 2±0.5 h.

[0018] The betaine-loaded composite hydrogel prepared by the method can be used in the preparation of functional foods, natural pigment delivery systems, or antioxidant active packaging.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] (1) This invention combines protein nanofiber reinforcement with enzymatic covalent crosslinking technology to prepare a composite hydrogel with a highly dense three-dimensional network structure and high mechanical strength. OAFs, as a nanoskeleton, form covalent bonds with the gelatin matrix through TGase mediation, which significantly improves the mechanical strength and thermal stability of the material and overcomes the defects of single gelatin gels being fragile and having poor thermal stability.

[0021] (2) The composite hydrogel prepared by this invention utilizes the abundant hydrophobic microdomains of OAFs to provide an excellent physical shielding environment for betalains. Experimental results show that the system can achieve an encapsulation rate of over 90% for betalains, and after 30 days of storage at room temperature and away from light, the retention rate of betalains is significantly higher than that of traditional single-matrix gels, effectively extending the shelf life of natural pigments.

[0022] (3) Although it is generally believed that a higher cross-linking density is more conducive to material encapsulation, this invention has found that the introduction of an appropriate amount of OAFs combined with moderate enzyme cross-linking can achieve the best sustained-release effect. This is because the aspect ratio advantage of OAFs and the site-directed cross-linking of TGase produce a synergistic effect, which, while ensuring the integrity of the gel structure, forms a microenvironment that is more conducive to protecting the active center of betalain, thus achieving zero release in simulated gastric juice and uniform sustained release in intestinal juice.

[0023] (4) This invention uses food-derived proteins (gelatin, egg white protein) as the main raw materials and adopts a mild enzymatic reaction instead of traditional chemical cross-linking agents. The entire preparation process is green, safe and non-toxic. This not only realizes the high-value utilization of biological macromolecules, but also the product has excellent biocompatibility, which fully meets the strict requirements of the food industry for natural and healthy delivery carriers. Attached Figure Description

[0024] Figure 1 This is a diagram showing the self-assembly kinetics of ovalbumin amyloid filaments (OAFs) of the present invention; where A is the ThT fluorescence spectrum of OAFs at different self-assembly times, and B is the kinetic curve of the highest fluorescence intensity changing with self-assembly time and the related fitting parameter table.

[0025] Figure 2 The images show the infrared spectra of the composite hydrogels in the embodiments and comparative examples of the present invention. GH represents the uncrosslinked control group, and GH-TG-2, GH-TG-4, GH-TG-6, and GH-TG-8 correspond to experimental groups with different amounts of OVFs added.

[0026] Figure 3 The bar chart shows the degree of crosslinking of the composite hydrogels in the embodiments and comparative examples of the present invention; it illustrates the effect of different amounts of OVFs added on the degree of crosslinking of the composite hydrogel system.

[0027] Figure 4 The rheological frequency scan diagrams of the composite hydrogels in the embodiments and comparative examples of the present invention show the changes in storage modulus (G′) and loss modulus (G′′) of each sample in the angular frequency range of 0.1-10 rad / s.

[0028] Figure 5 The bar chart shows the textural properties of the composite hydrogels in the embodiments and comparative examples of the present invention; it illustrates the effect of different OVF addition amounts on the hardness and springiness of the composite hydrogels.

[0029] Figure 6 The graphs show the cumulative release curves of betaine in the drug-loaded hydrogels of the present invention and comparative examples in a simulated gastrointestinal environment; they demonstrate the dynamic delivery behavior of betaine by composite hydrogels with different degrees of crosslinking in three stages: simulated saliva (SSF), simulated gastric juice (SGF), and simulated intestinal juice (SIF).

[0030] Figure 7 The graph shows the change in betalain loss rate of the drug-loaded hydrogels in the embodiments and comparative examples of the present invention during storage; it also shows the effect of different OVF addition amounts on the chemical stability of betalain in the composite hydrogel during a 28-day storage period.

[0031] Figure 8 The bar chart shows the water-holding capacity of the composite hydrogels in the embodiments and comparative examples of the present invention; it illustrates the effect of different ovalbumin amyloid fibrous (OAFs) addition amounts on the water-holding capacity of the composite hydrogel system.

[0032] Figure 9 The bar chart shows the bioaccessibility of the drug-loaded hydrogels in the embodiments and comparative examples of the present invention; it also shows the effect of different ovalbumin amyloid fiber (OAFs) addition amounts on the bioavailability of betalains in the composite hydrogel.

[0033] Figure 10 This is a SEM image of the composite hydrogel from Example 1.

[0034] Figure 11 This is a SEM image of the composite hydrogel from Example 2.

[0035] Figure 12 This is a SEM image of the composite hydrogel from Example 3.

[0036] Figure 13 This is a SEM image of the composite hydrogel from Example 4.

[0037] Figure 14 This is a SEM image of the composite hydrogel in Comparative Example 1.

[0038] Figure 15 This is a SEM image of the composite hydrogel in Comparative Example 2. Detailed Implementation

[0039] The present invention will be further described in detail below with reference to embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto. All raw materials involved in the present invention can be purchased directly from the market. For process parameters not specifically specified, conventional techniques can be referred to.

[0040] Preparation of ovalbumin amyloid fibrous (OAFs) dispersion: 1 g of ovalbumin was dissolved in 100 ml of water to prepare an ovalbumin solution with a concentration of 1 wt%. The pH was adjusted to 2 with hydrochloric acid, and the mixture was stirred at 90 °C for 12 h. After the reaction was completed, the mixture was immediately cooled in an ice bath to obtain the ovalbumin amyloid fibrous (OAFs) dispersion, which was then stored at 4 °C for later use.

[0041] Example 1

[0042] Weigh 10.0 g of gelatin powder and dissolve it in 100 mL of ultrapure water. Stir mechanically in a 50°C constant temperature water bath until completely dissolved. Add 200 mL of a 1.0 wt% OAFs dispersion to the above solution at a mass ratio of gelatin to ovalbumin amyloid fibrous (OAFs) dispersion of 10:2. Stir continuously for 30 min at 30°C and 1000 rpm until homogeneous to obtain the composite matrix solution.

[0043] 0.02 g of betaine solid was added to the composite matrix solution and stirred in the dark until completely dissolved. The cross-linking reaction was carried out in situ at 45 °C for 2 h. After the reaction was completed, the reaction solution was quickly injected into a mold and cooled and set at 4 °C for 12 h to obtain the betaine-loaded modified gelatin composite hydrogel, named GH-TG-0, abbreviated as GH.

[0044] Example 2

[0045] Weigh 10.0 g of gelatin powder and dissolve it in 100 mL of ultrapure water. Stir mechanically in a 50°C constant temperature water bath until completely dissolved. Add 200 mL of a 1.0 wt% OAFs dispersion to the above solution at a mass ratio of gelatin to ovalbumin amyloid fibrous (OAFs) dispersion of 10:2. Stir continuously for 30 min at 30°C and 1000 rpm until homogeneous to obtain the composite matrix solution.

[0046] 0.05 g of betaine solid was added to the composite matrix solution and stirred in the dark until completely dissolved. Then, transglutaminase (TGase) was added at a ratio of 40 U / g gelatin, and the mixture was subjected to in-situ crosslinking reaction at 50 °C for 2 h. After the reaction was complete, the reaction solution was quickly injected into a mold and cooled and set at 4 °C for 12 h to obtain the betaine-loaded modified gelatin composite hydrogel, named GH-TG-2.

[0047] Example 3

[0048] Weigh 10.0 g of gelatin powder and dissolve it in 100 mL of ultrapure water. Stir mechanically in a 50°C constant temperature water bath until completely dissolved. Add 400 mL of a 1.0 wt% OAFs dispersion dropwise to the above solution at a gelatin to ovalbumin amyloid fibrous (OAFs) mass ratio of 10:4. Stir continuously for 30 min at 30°C and 1000 rpm until homogeneous to obtain the composite matrix solution.

[0049] 0.05 g of betaine solid was added to the composite matrix solution and stirred in the dark until completely dissolved. Then, transglutaminase (TGase) was added at a ratio of 40 U / g gelatin, and the mixture was subjected to in-situ crosslinking reaction at 50 °C for 2 h. After the reaction was complete, the reaction solution was quickly injected into a mold and cooled and set at 4 °C for 12 h to obtain the betaine-loaded modified gelatin composite hydrogel, named GH-TG-4.

[0050] Example 4

[0051] Weigh 10.0 g of gelatin powder and dissolve it in 100 mL of ultrapure water. Stir mechanically in a 50°C constant temperature water bath until completely dissolved. Add 600 mL of a 1.0 wt% OAFs dispersion dropwise to the above solution at a gelatin to ovalbumin amyloid fibrous (OAFs) mass ratio of 10:6. Stir continuously for 30 min at 30°C and 1000 rpm until homogeneous to obtain the composite matrix solution.

[0052] 0.05 g of betaine solid was added to the composite matrix solution and stirred in the dark until completely dissolved. Then, transglutaminase (TGase) was added at a ratio of 40 U / g gelatin, and the mixture was subjected to in-situ crosslinking reaction at 50 °C for 2 h. After the reaction was complete, the reaction solution was quickly injected into a mold and cooled and set at 4 °C for 12 h to obtain the betaine-loaded modified gelatin composite hydrogel, named GH-TG-6.

[0053] Example 5

[0054] Weigh 10.0 g of gelatin powder and dissolve it in 100 mL of ultrapure water. Stir mechanically in a 50°C constant temperature water bath until completely dissolved. Add 800 mL of a 1.0 wt% OAFs dispersion dropwise to the above solution at a gelatin to ovalbumin amyloid fibrous (OAFs) mass ratio of 10:8. Stir continuously for 30 min at 30°C and 1000 rpm until homogeneous to obtain the composite matrix solution.

[0055] 0.05 g of betaine solid was added to the composite matrix solution and stirred in the dark until completely dissolved. Then, transglutaminase (TGase) was added at a ratio of 40 U / g gelatin, and the mixture was subjected to in-situ crosslinking reaction at 50 °C for 2 h. After the reaction was complete, the reaction solution was quickly injected into a mold and cooled and set at 4 °C for 12 h to obtain the betaine-loaded modified gelatin composite hydrogel, named GH-TG-8.

[0056] Example 6

[0057] Weigh 10.0 g of gelatin powder and dissolve it in 100 mL of ultrapure water. Stir mechanically in a 50°C constant temperature water bath until completely dissolved. Add 200 mL of a 1.0 wt% OAFs dispersion dropwise to the above solution at a gelatin to ovalbumin amyloid fibrous (OAFs) mass ratio of 10:2. Stir continuously for 30 min at 30°C and 1000 rpm until homogeneous to obtain the composite matrix solution.

[0058] 0.05 g of betaine solid was added to the composite matrix solution and stirred in the dark until completely dissolved. Then, transglutaminase (TGase) was added at a ratio of 10 U / g gelatin, and the mixture was subjected to in-situ crosslinking reaction at 50 °C for 2 h. After the reaction was complete, the reaction solution was quickly injected into a mold and cooled and set at 4 °C for 12 h to obtain the betaine-loaded modified gelatin composite hydrogel.

[0059] Example 7

[0060] Weigh 10.0 g of gelatin powder and dissolve it in 100 mL of ultrapure water. Stir mechanically in a 50°C constant temperature water bath until completely dissolved. Add 200 mL of a 1.0 wt% OAFs dispersion dropwise to the above solution at a gelatin to ovalbumin amyloid fibrous (OAFs) mass ratio of 10:2. Stir continuously at 1000 rpm for 30 min until homogeneous to obtain the composite matrix solution.

[0061] 0.05 g of betaine solid was added to the composite matrix solution and stirred in the dark until completely dissolved. Then, transglutaminase (TGase) was added at a ratio of 10 U / g gelatin, and the mixture was subjected to an in-situ crosslinking reaction at 55 °C for 0.5 h. After the reaction was complete, the reaction solution was quickly injected into a mold and cooled and set at 4 °C for 12 h to obtain the betaine-loaded modified gelatin composite hydrogel.

[0062] Example 8

[0063] Weigh 10.0 g of gelatin powder and dissolve it in 100 mL of ultrapure water. Stir mechanically in a 50°C constant temperature water bath until completely dissolved. Add 200 mL of a 1.0 wt% OAFs dispersion dropwise to the above solution at a gelatin to ovalbumin amyloid fibrous (OAFs) mass ratio of 10:2. Stir continuously at 1000 rpm for 30 min until homogeneous to obtain the composite matrix solution.

[0064] 0.1 g of betaine solid was added to the composite matrix solution and stirred in the dark until completely dissolved. Then, transglutaminase (TGase) was added at a ratio of 10 U / g gelatin, and the mixture was subjected to an in-situ crosslinking reaction at 55 °C for 0.5 h. After the reaction was complete, the reaction solution was quickly injected into a mold and cooled and set at 4 °C for 12 h to obtain the betaine-loaded modified gelatin composite hydrogel.

[0065] Comparative Example 1

[0066] Weigh 10.0 g of gelatin powder and dissolve it in 100 mL of ultrapure water. Stir mechanically in a 50°C constant temperature water bath until completely dissolved. Add 0.05 g of betaine solid to the composite matrix solution and stir in the dark until completely dissolved. Then, quickly pour the reaction solution into a mold and cool and set at 4°C for 12 h to obtain betaine-loaded gelatin hydrogel.

[0067] Comparative Example 2

[0068] Weigh 10.0 g of gelatin powder and dissolve it in 100 mL of ultrapure water. Stir mechanically in a 50°C constant temperature water bath until completely dissolved. Add 600 mL of a 1.0 wt% OAFs dispersion dropwise to the above solution at a gelatin to ovalbumin amyloid fibrous (OAFs) mass ratio of 10:6. Stir continuously for 30 min at 30°C and 1000 rpm until homogeneous to obtain the composite matrix solution.

[0069] 0.05 g of betaine solid was added to the composite matrix solution and stirred in the dark until completely dissolved. The reaction solution was then quickly poured into a mold and cooled and set at 4 °C for 12 h to obtain a composite hydrogel loaded with betaine.

[0070] Comparative Example 3

[0071] Weigh 10.0 g of gelatin powder and dissolve it in 100 mL of ultrapure water. Stir mechanically in a 50°C constant temperature water bath until completely dissolved. Add 600 mL of a 1.0 wt% untreated raw ovalbumin solution to the above solution at a gelatin to ovalbumin mass ratio of 10:6. Stir continuously for 30 min at 30°C and 1000 rpm until homogeneous to obtain the composite matrix solution.

[0072] 0.05 g of betaine solid was added to the composite matrix solution and stirred in the dark until completely dissolved. Then, transglutaminase (TGase) was added at a ratio of 40 U / g gelatin, and the mixture was subjected to an in-situ crosslinking reaction at 50 °C for 2 h. After the reaction was complete, the reaction solution was quickly injected into a mold and cooled and set at 4 °C for 12 h to obtain the betaine-loaded modified gelatin composite hydrogel.

[0073] like Figure 10 As shown, the composite hydrogel of Example 1 exhibits a typical large-pore lamellar structure. The pore walls are smooth and sheet-like, and the three-dimensional network distribution is relatively loose and discontinuous, lacking the physical filling and skeletal support of nanofibers.

[0074] like Figure 11 As shown, the gel of Example 2 exhibits a large, irregular pore structure with thin pore walls, relatively loose three-dimensional network connections, and numerous large-scale micropores on its surface.

[0075] like Figure 12 As shown, the gel pore size of Example 3 is significantly reduced, the spatial network structure becomes more regular, and the pore distribution tends to be uniform, showing preliminary densification characteristics.

[0076] like Figure 13 As shown, the gel of Example 4 exhibits a highly dense and uniform honeycomb three-dimensional network structure with the smallest pore size and excellent connectivity, increased protein backbone thickness, and the strongest structural stability.

[0077] like Figure 14 As shown, the gel of Comparative Example 1 exhibits an extremely irregular large-pore structure with extremely thin and uneven pore walls, poor network connectivity, and a structure that is obviously loose and brittle.

[0078] like Figure 15 As shown, the gel structure of Comparative Example 2 is disordered. Although a small amount of fibrous material can be seen, the overall network is broken and collapsed, lacking continuous and complete honeycomb pores, which proves that simple physical mixing is difficult to form a stable supporting skeleton.

[0079] Thionine (Tht) fluorescence analysis of OAFs

[0080] The self-assembly process of ovalbumin under acid-heat conditions was monitored using thioflavin T (ThT) fluorescent dye, and the results are as follows: Figure 1 As shown, ThT can specifically bind to co-crosslinked β-sheet structures in proteins, resulting in significant fluorescence enhancement.

[0081] Depend on Figure 1 As shown in Figure A, the fluorescence signal of the system was extremely weak in the unheated stage (Blank and 0 min). With the extension of heat treatment time, the fluorescence emission peak intensity at 485 nm showed a significant upward trend, indicating that the secondary structure of the ovalbumin molecule was rearranged and successfully assembled into a fibrous structure with typical amyloid characteristics.

[0082] Depend on Figure 1 The kinetic fitting curves of -B show that the assembly process of OAFs has obvious stages: within the first 10 minutes of the reaction, the fluorescence intensity increases exponentially, indicating that this stage is the rapid growth period of the fibers; after about 15 minutes, the fluorescence intensity gradually flattens and enters a plateau phase, indicating that the protein molecules in the system have basically completed self-assembly. Logistic model fitting confirms that the preparation process has good kinetic controllability. In summary, this invention successfully prepared high aspect ratio ovalbumin amyloid fibers via acid-thermal treatment, providing a high-quality nano-reinforcing phase for the subsequent construction of high-performance composite hydrogels.

[0083] GH-TG Infrared Spectroscopy Analysis

[0084] Depend on Figure 2 It can be seen that with the increase of TGase addition, the characteristic absorption peak positions of each sample (GH-TG-2 to GH-TG-8) are basically consistent with those of the GH control group, indicating that the enzymatic cross-linking process did not significantly change the protein backbone structure of gelatin and ovalbumin amyloid fibers. The dashed line in the figure indicates 1640 cm⁻¹. -1 The slight differences in peak intensity and changes in peak shape reflect the influence of TGase-catalyzed ϵ-(γ-glutamyl)lysine isopeptide bonds on the secondary structure of proteins, proving that TGase successfully induced covalent cross-linking between molecules within the complex system, thereby constructing a more stable three-dimensional network structure.

[0085] Crosslinking degree of composite hydrogels

[0086] The degree of crosslinking of the composite hydrogel was calculated by measuring the residual free amino content in the system, and the results are as follows: Figure 3 As shown. By Figure 3It was found that the crosslinking degree of the control group (GH) without added glutamine transaminase (TGase) was close to 0, indicating that in the absence of the enzyme, there was only a weak physical interaction between gelatin and ovalbumin amyloid fibers, making it difficult to form a stable covalent network. With increasing TGase addition, the crosslinking degree of the composite hydrogel showed a significant gradient increase: when the TGase addition increased from TG-2 to TG-8, the crosslinking degree significantly increased from approximately 18% to about 64%. This result quantitatively proves that TGase successfully catalyzed the formation of ε-(γ-glutamyl)lysine covalent isopeptide bonds between gelatin molecular chains and between gelatin and ovalbumin amyloid fibers. The significant increase in crosslinking degree means that the compactness of the three-dimensional network structure inside the hydrogel is greatly enhanced. This highly crosslinked covalent network not only provides strong mechanical support for the composite system but also facilitates spatial confinement, thereby firmly embedding betaine molecules within the network and reducing their migration and degradation under external environmental stimuli. This is highly consistent with the increased hardness observed in subsequent textural analysis and the excellent sustained-release performance demonstrated in simulated gastrointestinal release experiments.

[0087] Rheological property analysis of composite hydrogels

[0088] The dynamic viscoelastic characteristics of the composite hydrogel were characterized using a rheometer, and the frequency scanning results are as follows: Figure 4 As shown. By Figure 4 It was found that within the tested angular frequency range (0.1-10 rad / s), the storage modulus of all samples (GH, GH-TG-2, GH-TG-4, GH-TG-6) was significantly higher than their loss modulus, and the frequency dependence was weak, exhibiting a relatively stable plateau region. This indicates that the composite system has successfully formed a stable three-dimensional cross-linked network, exhibiting typical solid-like elastic behavior. With the increase of the amount of transglutaminase (TGase) added, the storage modulus of the samples showed a significant gradient increase. Among them, the GH-TG-6 group had the highest value, significantly better than the uncross-linked GH control group. This phenomenon proves that the covalent isopeptide bonds generated by TGase catalysis significantly increased the cross-linking density inside the hydrogel, enhancing the rigidity and deformation resistance of the network structure. At the same time, the low loss tangent value further illustrates that the composite hydrogel has excellent structural integrity and kinetic stability. This tough gel skeleton can provide a stable physical barrier for the betaine embedded within, which is highly consistent with its excellent sustained-release performance and processing stability results.

[0089] Analysis of the textural properties of composite hydrogels

[0090] The mechanical properties of the composite hydrogel were characterized using a texture analyzer, and the results are as follows: Figure 5 As shown. By Figure 5It was found that with the increase of transglutaminase (TGase) addition, the hardness and elasticity of the composite hydrogel showed a significant upward trend: the hardness of the uncrosslinked control sample (GH) was approximately 500 g. With the increase of enzyme amount, the hardness gradually increased from approximately 560 g for GH-TG-2 to over 640 g for GH-TG-8. This significant increase in hardness is attributed to the fact that the covalent isopeptide bonds formed by TGase catalyze significantly increased the network density, and together with the nano-reinforcing effect of ovalbumin amyloid filaments (OAFs), a higher-strength three-dimensional network framework was constructed. The elasticity of the composite hydrogel showed a more significant increase with the increase of enzyme amount. The elasticity of the control GH was relatively low (approximately 0.2), while with the deepening of crosslinking, the elasticity in the GH-TG-8 group increased to approximately 0.65.

[0091] Texture analysis results show that this invention significantly improves the mechanical processing properties of gelatin-based hydrogels through the synergistic effect of enzymatic crosslinking and nanofiber reinforcement. Higher hardness endows the carrier with excellent physical stability, making it less prone to breakage during processing and transportation; while improved elasticity means the gel network has stronger structural recovery capabilities, better resisting external mechanical stress. This robust mechanical property provides excellent physical protection for the internally embedded betaine, forming the basis for its stable delivery.

[0092] In vitro simulated gastrointestinal delivery performance analysis of composite hydrogel

[0093] The encapsulation, protection, and sustained release capabilities of the composite hydrogel for betaine were evaluated using an in vitro simulated digestion model. The results are as follows: Figure 6 As shown. By Figure 6It was found that all samples exhibited distinct phased release characteristics during simulated digestion: Gastric protection phase (SGF, 0-4 h): The control group (GH-TG-0) without added glutamine transaminase (TGase) showed the highest cumulative release rate in the gastric environment, reaching approximately 60% at 4 h. In contrast, the early release of betaine was significantly inhibited with increasing TGase addition. Among them, the GH-TG-8 group, with the highest degree of cross-linking, showed the lowest release rate (approximately 45%) in the gastric fluid phase. This demonstrates that the dense three-dimensional network composed of enzymatically covalently cross-linked and ovalbumin amyloid fibers can effectively shield against gastric acid penetration, reducing premature loss of betaine in the stomach and thus increasing its effective dose reaching the intestines. Intestinal sustained-release phase (SIF, 4-12 h): After entering the simulated intestinal fluid, the release rates of each group tended to stabilize, exhibiting typical sustained-release kinetics. Notably, the cumulative release rate of the control group GH-TG-0 tended to stagnate in the later stages, while the cumulative release rate of the cross-linked groups (such as GH-TG-8) eventually reached nearly 80%. These experimental results quantitatively demonstrate that the composite hydrogel described in this invention not only significantly improves the stability of betaine in the stomach but also achieves efficient and sustained release of functional components in the intestinal environment. This performance improvement is attributed to the high cross-linking density induced by TGase and the nano-confinement effect of amyloid fibers, which synergistically construct a protective matrix with high structural integrity. This is highly consistent with the structural reinforcement characteristics observed in previous infrared, cross-linking degree, and texture tests, demonstrating the enormous application potential of this system in the delivery of functional foods and biopharmaceuticals.

[0094] Storage stability analysis of composite hydrogels

[0095] The protective effect of the composite system during long-term storage was evaluated by periodically measuring the residual content of betaine in the hydrogel and calculating its loss rate. The results are as follows: Figure 7 As shown. By Figure 7It was found that during the 28-day storage observation period, the betaine loss rate of all samples showed an increasing trend to varying degrees with the extension of storage time. This is attributed to the spontaneous oxidation and degradation of natural pigments under the influence of environmental factors. Comparing different groups, it was observed that the protective behavior of composite hydrogels with different cross-linking densities on betaine differed significantly: under the experimental conditions, the control group GH-TG-0 showed a relatively low loss rate; while with the increase of TGase addition, the betaine loss rate showed a gradient increasing trend, with the GH-TG-8 group showing the highest loss rate at 28 days. This indicates that although high-density enzymatic covalent cross-linked networks have excellent controlled-release capabilities under simulated digestion conditions, during long-term storage, overly dense protein networks may accelerate betaine degradation to some extent by altering the osmotic pressure of the microenvironment or intermolecular interactions, or affect the extraction and detection rate of pigment molecules. Although the highly cross-linked group exhibits a higher loss rate in storage stability, the composite hydrogel system described in this invention still possesses significant technical advantages and application value in the field of precise release of functional foods, thanks to its excellent targeted delivery and sustained-release properties in the gastrointestinal tract. By adjusting the amount of TGase added, a balance can be achieved between storage stability and release kinetics, thereby meeting different application requirements.

[0096] Analysis of the water-holding capacity of composite hydrogels

[0097] Depend on Figure 8 It can be seen that with the increase of OAFs addition, the water-holding capacity (WHC) of the composite hydrogel shows a significant upward trend, increasing from about 90.5% in the control group (GH) to over 94.5% in the GH-TG-8 group. This further demonstrates that the dense three-dimensional network formed by OAFs nanofibers and gelatin can firmly lock water molecules in the gel matrix through stronger spatial confinement effects and hydrogen bonding, thereby endowing the material with better structural stability.

[0098] Bioaccessibility analysis of composite hydrogels

[0099] Depend on Figure 9 It is known that free betalains (Bet) have a bioavailability of only about 38.4% due to their sensitivity to the digestive tract environment. Encapsulating them in hydrogels significantly improves their bioavailability. Compared to 56.2% for single gelatin gel (GH), the bioavailability of the composite hydrogel group shows a clear gradient increase with increasing OAFs (organic acid oxidases). The GH-8 group exhibits the highest bioavailability, exceeding 82%. This result is consistent with simulated gastrointestinal delivery performance tests (…). Figure 6This is consistent with the findings. This indicates that the dense protective network constructed by protein nanofibers in this invention not only effectively reduces the degradation loss of betaine in the stomach (SGF) but also achieves a more stable and efficient release in the intestine (SIF), thereby significantly improving the actual bioavailability of betaine in vivo.

Claims

1. A method for preparing a betaine-loaded composite hydrogel, characterized in that, Includes the following steps: (1) The ovalbumin solution was heat-treated under acidic conditions to induce its self-assembly into an ovalbumin amyloid cellulose dispersion; (2) Mix the ovalbumin amyloid fiber dispersion with the gelatin solution and stir to dissolve to obtain a matrix mixture; (3) Add betaine and transglutaminase to the obtained matrix mixture, react at 37~55℃ for 0.5~3h, and then cool and fix to obtain betaine-loaded composite hydrogel.

2. The preparation method according to claim 1, characterized in that, The mass ratio of gelatin to ovalbumin amyloid dispersion in step (2) is 10:1-1:1, the concentration of ovalbumin amyloid fiber dispersion is 0.5~2.0 wt%, and the concentration of gelatin solution is 0.05~0.15 g / mL.

3. The preparation method according to claim 2, characterized in that, The mass ratio of gelatin to ovalbumin amyloid cellulose dispersion in step (2) is 5:1-5:4; the pH of ovalbumin amyloid cellulose dispersion is 2.0±0.

5.

4. The preparation method according to claim 1, 2, or 3, characterized in that, The ovalbumin amyloid fibers were prepared by the following method: The pH of an ovalbumin solution with a concentration of 1.0~2.0 wt% was adjusted to 1.5~2.5 with hydrochloric acid, and the mixture was stirred at a constant temperature of 80~95℃ for 12~24h.

5. The preparation method according to claim 1, 2, or 3, characterized in that, The amount of transglutaminase added in step (3) is 10~50 U / g, based on the weight of gelatin.

6. The preparation method according to claim 5, characterized in that, The amount of transglutaminase added in step (3) is 20~40 U / g, based on the weight of gelatin.

7. The preparation method according to claim 5, characterized in that, The amount of betalain added is 0.1~0.5 wt% of the gelatin mass.

8. The preparation method according to claim 1, 2, or 3, characterized in that, The stirring and dissolving conditions in step (2) are mechanical stirring at 500-1000 rpm for 15-30 min at 40-60℃; the reaction temperature in step (3) is 45-50℃ and the time is 2±0.5h.

9. The betaine-loaded composite hydrogel prepared by the method according to any one of claims 1-8.

10. The use of the betaine-loaded composite hydrogel of claim 9 in the preparation of functional foods, natural pigment delivery systems, or antioxidant active packaging.

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